According to the invention P-tubulin., as well as a new polypeptide present in pancreatic xcex2-cells, have been identified as molecular targets for sulfonylurea compounds. These findings enable for the identification of new insulin secretagogues. The invention thus relates to the use of sulfonylurea compounds, such as e.g. glibenclamide, in methods for identification of compounds binding the new polypeptide or tubulin, or stimulating tubulin polymerization and/or turnover, said compounds thereby stimulating insulin secretion.
Diabetes mellitus is a chronic disease affecting approximately 3-5% of the Swedish population and it is associated with a variety of severe late complications leading to enhanced morbidity and mortality. Thus, this disease markedly compromises the health and life quality of affected individuals and consumes.a substantial amount of the health care budget.
Hyperglycemia is a major risk factor for the development of the specific diabetes-associated complications and probably also for the increased risk of cardiovascular disease. Early intervention, which requires diagnosis in the prediabetic state, is expected to contribute to a reduction of the diabetic complications. Hence, there is an urgent need for precise methods in the early detection of individuals at risk for the development of diabetes. Furthermore, there is a need for the design of new concepts and drugs for prevention and treatment of diabetes. A better understanding of the biochemical, cellular, genetic and molecular basis for the development of hyperglycemia and the effect of hyperglycemia on both release and action of insulin will pave the way for new drugs and future development of gene therapy in the treatment of diabetes. A defective insulin release is a characteristic of non-insulin dependent diabetes mellitus (NIDDM) and to some extent also to the early phase of insulin-dependent diabetes mellitus (IDDM).
An initial step in the xcex2-cell stimulus-secretion coupling is metabolism of glucose, resulting in the formation of ATP. ATP closes the ATP-regulated K+-channels (KATP channels), resulting in plasma membrane depolarization, opening of voltage-gated L-type Ca2+-channels and increase in cytoplasmic free Ca2+ concentration, [Ca2+]i. The KATP channel is composed of at least two components: a sulfonylurea receptor (SUR) and an inward rectifier potassium channel protein (Aguilar-Bryan, L. et al. (1995) Science 268: 423-426). The binding of sulfonylureas (SU) to SUR1, the xcex2-cell variant of SUR, results in the closure of the KATP channels and thereby insulin release in the xcex2-cell. Sulfonylurea compounds, such as glibenclamide (glyburide; 5-Chloro-N-[2-[4-[[[(cyclohexylamino) carbonyl]amino]sulfonyl]phenyl]ethyl]-2-methoxybenzamide, CAS: [10238-21-8]) have been used in the treatment of NIDDM (for a review, see Luzi, L. and Pozza, G. (1997) Acta Diabetol. 34, 239-244).
Sulfonylurea compounds are capable of promoting insulin secretion even in the absence of changes in membrane potential and intracellular calcium. It has been shown that sulfonylurea compounds directly promote exocytosis of insulin (Eliasson L. et al. (1996) Science 271: 813-815; Flatt et al. (1994) Diabete et Metabolisme 20: 157-162). This effect is dependent on protein kinase C and is observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics. It has been suggested that 80-90% of the SU binding proteins is localized to intracellular membranes, including those of the secretory granules (Ozanne S. E. et al. (1995) Diabetologia 38, 277-282). The molecular mechanism underlying this direct effect of sulfonylureas on insulin exocytosis is not known.
Consequently, there is a need for identification of new intracellular targets that could explain the effect of sulfonylureas on insulin exocytosis via a pathway independent of the sulfonylurea receptor (SUR-1) and the ATP-regulated K+-channel. Such targets could be utilized in methods for the identification of new compounds stimulating insulin secretion.
The involvement of the microtubular network in glucose-induced insulin secretion has been suggested (Lacy, P. E. et al. (1968) Nature 219, 1177-1179). The hypothesis was based on the decreased insulin secretion observed in the presence of drugs that inhibit polymerization of the tubulin heterodimer to form microtubules. Subsequent studies revealed that the reduced secretion was observed both in the presence of microtubule stabilizers (e.g. D2O and ethanol) and destabilizers (e.g. colchicine and vincristine) (Malaisse, W. J. et al. (1970) Diabetologia 6, 683; Malaisse, W. J. et al. (1971) Diabetes 20, 257-265). It was also shown that the observed effect did not result from an altered insulin production or calcium uptake, demonstrating that it was the transport and/or secretion of insulin containing granules that was not functional. Following these results a model of insulin secretion was presented in which the second sustained phase of secretion depends on the directional transport of insulin granules along the microtubular network (Malaisse, W. J. et al. (1974) Eur. J. Clin. Invest. 4, 313-318).
A number of reports have since confirmed the importance of the microtubular system in insulin secretion (for a review, see Howell, S. L. and Tyhurst, M. (1986) Diabetes/Metabolism Reviews, 2, 107-123). These studies revealed that the integrity and/or the dynamic equilibrium (treadmilling as well as phases of microtubule growth and shortening) of the microtubules are essential for glucose-stimulated insulin secretion. Of interest from a pharmaceutical viewpoint is to identify agents that act on the microtubule network to specifically enhance insulin secretion. This task is not trivial as the microtubular network is generally involved in secretory processes in several endocrine organs and glands (see e.g. Poisner, A. M. and Bernstein, J. (1971) J. Pharmacol. Exp. Ther. 177, 102-108; Neve, P. et al. (1970) Exp. Cell Res. 63, 457-460; Williams, J. A. and Wolff, J. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 1901-1908; Kraicer, J. and Milligan, J. V. (1971) Endocrinology 89, 408-412) and hence not expected to be a suitable target for a specific insulin secretagogue.